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Oligonucleotide conjugated antibody strategies for cyclic immunostaining.
- Source :
-
Scientific Reports . 12/13/2021, Vol. 11 Issue 1, p1-18. 18p. - Publication Year :
- 2021
-
Abstract
- A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 20452322
- Volume :
- 11
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Scientific Reports
- Publication Type :
- Academic Journal
- Accession number :
- 154097280
- Full Text :
- https://doi.org/10.1038/s41598-021-03135-9